超声介导转铁蛋白修饰共载表阿霉素和薯蓣皂苷脂质体微泡复合物双靶向给药系统的构建及其逆转多药耐药研究

Multidrug resistance (MDR) is one of the major obstacles to the successful cancer chemotherapy. It is known that MDR is frequently associated with upregulation of the P-glycoprotein (P-gp), an efflux pump that reduces intracellular drug concentration. To overcome MDR, the general strategy has been to co-administer anti-cancer drugs with chemical inhibitors of P-gp. Unfortunately, this approach has not yet reached clinical success due to the complex array of drug toxicity, altered pharmacokinetics and adverse drug interactions. In this study, transferrin-transferrin receptor induced active targeting technology in combination with ultrasound triggered local targeting technology is used for the first time to prepare ultrasound triggered transferrin conjugated epirubicin and dioscin co-loaded dual tumor targeting liposome-microbubble-complexes drug delivery system (Tf-EPI/Dioscin-LMC) by coupling drug containing liposomes to the surface of microbubbles. The purpose of this study is to investigate the effectiveness and possible mechanisms of the reversal of MDR phenotype in human breast cancer MCF-7/ADR cells by using ultrasound triggered Tf-EPI/Dioscin-LMC, and to clarify the ameliorating effects of ultrasound, transferrin, P-gp inhibitor dioscin and microbubbles in overcoming drug resistance as parts of Tf-EPI/Dioscin-LMC drug delivery system. The design of this formulation is aimed at improving cellular uptake of EPI, decreasing cellular efflux of EPI mediated by P-gp，increasing intracellular EPI accumulation, reducing cardiotoxicity of EPI by selective targeting of tumor cells, as well as overcoming P-glycoprotein-mediated MDR phenotype. The aim of this work is to develop and generate a targeted, effective and non-toxic novel strategy to eradicate the presence of MDR in cancer by pharmaceutical approach. This study provides important information to circumvent MDR and to enhance the therapeutic index of the chemotherapy.

多药耐药（MDR）是导致临床化疗失败的主要原因之一，目前认为P-糖蛋白（P-gp）过表达造成抗癌药物外排增加、药效降低，是耐药形成的主要原因。针对P-gp介导的MDR，单纯联合使用抗癌药物与P-gp抑制剂的传统逆转策略，由于逆转耐药效率低、毒副作用大而限制其临床应用。本研究将转铁蛋白-转铁蛋白受体介导的主动靶向释药技术与超声微泡定向释药技术相结合，通过脂质体与微泡的共价连接，首次构建超声介导转铁蛋白修饰的共载表阿霉素（EPI）和薯蓣皂苷脂质体微泡复合物双靶向给药系统，并阐明其逆转人乳腺癌MCF-7/ADR细胞多药耐药的分子作用机制，明确载药系统各组成部分在逆转MCF-7/ADR细胞多药耐药中的作用，旨在从药剂学入手，提高人乳腺癌耐药细胞对EPI的摄取，减少P-gp的外排，增加EPI在肿瘤细胞中的蓄积，力求寻找一种高效、低毒逆转肿瘤细胞多药耐药的新策略，为临床提高EPI肿瘤化疗效果提供依据。

本研究将转铁蛋白-转铁蛋白受体介导的主动靶向释药技术与超声微泡定向释药技术相结合，通过脂质体与微泡的共价连接，制备超声（US）介导转铁蛋白修饰的共载表阿霉素（EPI）和薯蓣皂苷脂质体微泡复合物双靶向给药系统（Tf-EPI/Dioscin-LMC），旨在从药剂学入手，力求寻找一种高效、低毒的能够逆转肿瘤细胞多药耐药的新策略。我们首先采用薄膜分散技术、聚碳酸酯膜挤出法及pH梯度主动载药技术制备共载EPI和薯蓣皂苷的脂质体（EPI/Dioscin-L）；然后利用亲和素-生物素桥将转铁蛋白修饰的EPI/Dioscin-L（Tf-EPI/Dioscin-L）与脂质微泡（Microbubbles）连接起来，成功构建了脂质体微泡复合物（Tf-EPI/Dioscin-LMC）。我们发现，Tf-EPI/Dioscin-LMC+US治疗能显著增强EPI对MCF-7/ADR 细胞的细胞毒性，表明Tf-EPI/Dioscin-LMC+US作为一种新型的药物传递系统，可以增强EPI对这些MDR细胞表型的作用。其作用机制可能包括以下几个方面：首先，Tf-EPI/Dioscin-LMC+US载药系统可能通过提高耐药细胞对药物的摄取，增加抗癌药物在肿瘤细胞中蓄积，从而逆转肿瘤细胞多药耐药；第二，可能通过抑制膜糖蛋白P-gp的表达，弱化P-gp介导的药物外排泵机制，进而增强药物对肿瘤细胞的杀伤作用；第三，可能通过诱导MCF-7/ADR细胞的凋亡，增强EPI对耐药细胞的毒性。此外，细胞内由于超声空化作用产生的大量活性氧对逆转MCF-7/ADR细胞的多药耐药过程中可能也会起到非常重要的作用。本研究尝试通过制剂学手段逆转肿瘤细胞对EPI的耐药现象，为临床提高EPI的化疗效果提供了新的思路。在国家自然科学基金资助下，已发表论文五篇，培养本科生两名。资助经费总额为17.9万元，总支出为16.4914万元，各项支出基本与预算相符，剩余经费计划用于本项目研究的后续支出。